P1370: A NOVEL GENE THERAPY VECTOR FOR BETA-HEMOGLOBINOPATHIES WITH A SELECTIVE EXPANSION CASSETTE
Authors: Alexandra Kirtsou, Aimilia Panagiotou, Kiriaki Paschoudi, Ioannis Ninos Vasiloudis, Haroula Piperidou, Evangelia Yannaki, Nikoletta Psatha
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Gene therapy has recently been established as an effective curative approach for a variety of genetic hematopoietic diseases,including beta-hemoglobinopathies.Despite the promising results in gene therapy trials for beta-thalassemia and sickle cell disease and although GVHD is being averted,due to autologous transplantation of ex vivo corrected hematopoietic stem cells(HSCs),there are several important limitations of the currently used gene therapy vectors minimizing the overall outcome of the approach.Among these,the relative inefficient transduction of long term HSCs especially in lower vector copy numbers(VCN) and the possibility of integration site-related gene silencing over time,insertional mutagenesis,have yet to be resolved. Aims: Scope of this study is to design and assess a new lentiviral vector for the gene therapy of beta-hemoglobinopathies.The novel vector comprises of an short-hairpin RNA(shRNA) targeting the HbF suppressor BCL11A,a new,compact,erythroid specific enhancer driving the transcription of the transgene,a recently identified insulator with enhancer-blocker and barrier activity and the F36VMpl selection cassette,a fusion protein,able to ignite a growth signal in response to a chemical inducer of dimerization.The addition of the new erythroid enhancer in combination with the dual insulator aim to improve the safety and long term efficacy profile of the vector and the incorporation of the selective expansion cassette aims to improve therapeutic outcomes in low VCNs. Methods: The shRNA-BCL11a-F36VMpl vector was constructed with the addition of a GFP reporter gene for normalization purposes. Viral titers of the new vector were compared to a similar control vector excluding the F36VMpl transgene.HUDEP-2 cells as well as human normal and sickle cell disease patient derived hematopoietic stem and progenitor cells(CD34+) were transduced at low MOIs and subsequently cultured in the presence or absence of AP20187.Transduction,selective proliferation and expression of γ-globin in erythroid cells was evaluated by flow cytometry and HPLC. Results: The addition of the selection transgene did not affect the produced viral titers(36x106 TU/ml vs 16x106 TU/ml,in the control vector respectively).In HUDEP-2 cells,the addition of AP20187 led to a significant(29%) increase in the frequency of transduced cells on day 12 of the culture compared to the minus-AP20187 condition(81%±0.45 vs 52%±1.57).In addition,the percentage of HbF+ cells was 35% in F36VMpl-cells while the corresponding percentage in untrasduced cells was 8%.The transduction rate in CD34+ cells ranged from 1-65%,depending on the MOI used.With the addition of AP20187 to the erythroid culture of CD34+ cells,an increase in the percentage of transduced GFP+ cells in the total population was observed,starting from a 3-fold and reaching a 8-fold increase at the lowest MOI.In addition,γ-globin/ β-globin ratio in both normal and SCD CD34+ cells was increased in the presence of AP20187(normal CD34+: 0,34 +AP20187 vs 0,19 -AP20187/SCD CD34+: 0,6 + AP20187 vs 0,41 -AP20187). Summary/Conclusion: We have so far shown that the shRNA-BCL11a-F36VMpl viral vector can achieve an erythroid-specific silencing of BCL11A with a subsequent activation of fetal hemoglobin and has the ability to selectively expand the corrected cell population.As it has been shown that AP20187-mediated activation of F36VMpl allows for progenitor cells to be dramatically expanded both in vitro and in vivo without toxic side effects,we theorize that this vector might overcome the inefficiency of ex vivo gene therapy correlated with low transducibility. Keywords: Lentiviral vector, Hemoglobinopathy, CD34+ cells, Gamma globin